Project Summary/Abstract: Early diagnosis and treatment of melanoma are essential to minimizing its morbidity and mortality given its very poor prognosis with median survival of less than 1 year. The presence of lymph node metastases is a vital prognostic predictor, and accurate identification is thus essential to accurately stage melanoma in its earlier phases. Sentinel lymph node (SLN) mapping procedures are currently limited by a lack of intraoperative visualization tools that can aid accurate determination of disease spread and delineate nodes from adjacent critical neural and vascular structures. Thus, newer, transformative and clinically translated surgical visualization tools are needed to reliably detect early-stage disease and offer improved sensitivity and accuracy in the molecular characterization of multiple cancer (and/or vital normal tissue) targets, including disease spread beyond the SLN, and the delineation of these from adjacent normal nerves. Ultrasmall (sub 10- nm) fluorescent core-shell silica nanoparticles modified by melanoma-targeting peptides and PET radiolabels (124I) represent one such platform. A first-in-human clinical trial based on an early generation ?v?3 integrin- targeting particle probe, Cornell dots (C dots), showed favorable pharmacokinetic (PK) signatures and bulk renal excretion in metastatic melanoma patients. Extended to SLN mapping studies in a spontaneous melanoma miniswine model, this dual-modality integrin-targeting particle was found to specifically detect, localize, and discriminate cancer-bearing lymph nodes of varying tumor burden by PET and image-directed intraoperative near-infrared (NIR) fluorescence imaging, the latter tool confirming optically-active nodal metastases by direct visualization and pathological correlation. In order to enhance and more accurately identify molecular cancer phenotypes in the intraoperative setting, we will utilize these integrin-targeting platforms in conjunction with newly developed and spectrally-distinct NIR CW800 dye-containing C' and much brighter alumina silicate particle products, AC' dots bearing peptides targeting a second well-established cancer marker, melanocortin-1 receptor (MC1-R), a promoter of melanoma cell growth. Clinically translated integrin-targeting platforms will be used to inform the development of these newer generation classes of MC1- R targeting C' and AC' dots. This proposal aims to: (1) develop and validate a series of CW800 dye-containing melanoma-targeting particles specific for MC1-R on the basis of design strategies utilized for fluorescent cRGDY-based platforms; (2) assess tumor-selective accumulations and PK profiles of hybrid 124I-?MSH-PEG- CW800-C' dot candidates in MC1-R-expressing xenografts, genetically engineered mouse models, and melanoma miniswine models; (3) develop spectrally-distinct NIR dye-containing products that allow for simultaneous differentiation of different pathological markers on diseased nodal tissue and normal tissue types for intraoperative optical imaging guidance; and (4) perform IND-enabling studies for clinical trial development. A longer-term objective of this work is to improve the efficiency of probe translation to the clinic by identifying and qualifying reliable biomarkers that can be validated in our future clinical trial designs with an emphasis on those for cancer staging.